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Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p 0.01) and improved markedly at 168 h in the spleen (p 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1 pg/μl for genomic DNA of tested bacteria and 10-4 to 10-5 pg/μl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Iridoviruses, especially megalocytiviruses, are related to severe disease resulting in high economic losses in the aquaculture industry worldwide. The ornamental fish industry has been affected severely due to Megalocytivirus infections. Megalocytivirus is a DNA virus that has three genera; including red sea bream iridovirus, infectious spleen and kidney necrosis virus, and turbot reddish body iridovirus. Megalocytivirus causes non-specific clinical signs in ornamental fish. Cell culture, histology, immunofluorescence test, polymerase chain reaction (PCR) assay, and loop-mediated isothermal amplification assay have been used to diagnose megalocytiviruses. Risk factors such as temperature, transportation (export and import), and life stages of ornamental fish have been reported for the previous cases due to Megalocytivirus infections. In addition, other prevention and control methods also have been practiced in farms to prevent Megalocytivirus outbreaks. This is the first review of megalocytiviruses in ornamental fish since its first detection in 1989. This review discusses the occurrences of Megalocytivirus in ornamental fish, including the history, clinical signs, detection method, risk factors, and prevention measures.

Co-infections with pathogenic microorganisms are common in aquaculture, resulting in more serious economic losses than single-pathogen infection. Infection of Aeromonas hydrophila (A. hydrophila) often occurs together with infectious spleen and kidney necrosis virus (ISKNV) in Chinese perch (Siniperca chuatsi) culture ponds. In this study, A. hydrophila and ISKNV were inoculated into Chinese perch to mimic individual infection, secondary infection, and mixed infection. The effects of concurrent infections on the susceptibility and the immune response of the host and changes in bacterial and viral load were studied. The results showed relatively complex interaction between ISKNV and A. hydrophila for different infection modes, acting in an antagonistic or synergistic manner. The experimental groups infected with a mixture of ISKNV and A. hydrophila showed higher mortality rate than groups infected with single-pathogen or secondary infection groups, suggesting a synergistic lethal effect of A. hydrophila and ISKNV co-infection. Serious clinical symptoms and obvious histopathological changes were observed in moribund fish under the mixed-infection condition. In addition, obviously higher mortalities were caused by secondary bacterial infections than the number caused by secondary viral infections. ISKNV-primary infection increased the mortality caused by secondary bacterial infections, but A. hydrophila-primary infection did not significantly increase the mortality caused by secondary viral infections. Co-infected fish showed high expression levels of IRF1, Mx, Viperin, Hepcidin, TNFα, and IL-1β mRNAs relative to the levels in healthy fish, which suggested that the co-infection of these two pathogens activated the host immune system and caused host inflammation. These results of infection with A. hydrophila and ISKNV provided the theoretical basis to analyze the pathogenic effects and interaction between pathogens, and could facilitate design of strategies for clinical prevention and control measures of outbreak of fulminant hemorrhagic disease and bacterial sepsis in Chinese perch.

Megalocytivirus of family Iridoviridae has been identified as a pathogen that caused the fatal systemic infection to lead to massive death of numerous fish species in both ornamental and food fish industries. It caused significant economic losses due to severe mortality of infected fish. Since 2011 in Indonesia, megalocytivirus in giant gourami (Osphronemus goramy) had been found as the main pathogen in some cases of giant gourami outbreak, especially in West Java, Central Java, and Bali. The aim of this study is to identify and characterize the pathogen known as a megalocytivirus infection in freshwater-cultured giant gourami. We identified the virus using universal and specific primers for megalocytivirus and infectious spleen and kidney necrosis virus (ISKNV). Sequencing and BLAST analysis were used to develop a phylogenetic tree. The result showed that phylogenetic analysis of major capsid protein (MCP) gene unveiled a new member of megalocytivirus, designated as giant gourami iridovirus (GGIV). GGIV-formed cluster belonged to ISKNV and has 100% homology to ISKNV complete genome. Artificial infection by intraperitoneal injection with supernatant homogenate from spleen and kidney of naturally infected fish showed 93% cumulative mortality in 12 days. Fish showed a clinical sign of infection as lethargic, loss of appetite, pale or darken body color, and hemorrhages. Internal organ on dead fish showed swollen spleen and kidney and also a pale liver. Quantitative PCR analysis on internal organs showed spleen had the highest viral DNA copy number followed by kidney, gill, and liver. Histopathological analysis showed many abnormally hypertrophied cells in spleen which is typical histopathological characteristic of megalocytivirus infection. In conclusion, GGIV belonged to ISKNV from genus megalocytivirus.

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture.

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.

Infectious spleen and kidney necrosis virus (ISKNV) resulted in severe systemic diseases with high morbidity and mortality in Siniperca chuatsi. Vaccination is the primary method for effective prevention and control of these diseases. The development of inactivated ISKNV vaccines made some progress, but the technique of quality evaluation is scarce. Herein, a measurement of the MCP (major capsid protein) antigen concentration for the inactivated ISKNV vaccine was developed by double-antibody sandwich ELISA. Firstly, mouse monoclonal antibodies against ISKNV particles and MCP were generated. Then, a double-antibody sandwich ELISA was developed using the monoclonal antibody 1C8 1B9 as the capture antibody and Biotin-3B12 6B3 as the detection antibody. A standard curve was generated using the MCP concentration versus OD value with the linear range of concentration of 4.69~300 ng/mL. The assay sensitivity was 0.9 ng/mL. The antigen content of three batches of inactivated ISKNV vaccines was quantitatively detected using the double-antibody sandwich ELISA. The results showed that MCP antigen contents of inactivated ISKNV vaccines were positively correlated with the viral titers. The newly established double-antibody sandwich ELISA provided a useful tool for the detection of antigen quality for ISKNV inactivated vaccines.

Megalocytivirus is a genus of piscine viruses that belongs to the Iridoviridae family, and this family includes red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and infectious spleen and kidney necrosis virus. RSIV causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) Korean aquaculture industry. The World Organization for Animal Health’s Office International des Epizooties has provided a manual for RSIV detection. However, distinguishing it from TRBIV has not been confirmed. In this study, a multiplex PCR method was established to detect two genotypes of Megalocytivirus, RSIV and TRBIV. New primer pairs were optimized for the PCR reaction. A mixture of one universal and two specific primer pairs could amplify three distinct products targeting ATPase, DPO, and LRP genes. The sensitivities of primer pairs were evaluated, showing a detection limit of 2.0 × 105 copies, using a plasmid construct, against the three target genes of Megalocytivirus. Moreover, specificity evaluation indicated that primer pairs were not subjected to interference by other virus strains. This evaluation of multiplex PCR showed that it can distinguish two genotypes of Megalocytivirus from 21 Korean Megalocytivirus isolates, including 20 RSIV and one TRBIV. Finally, we describe a new multiplex PCR method to detect different genotypes of Megalocytivirus simultaneously, which makes the diagnosis of viral diseases occurring in the Korean aquaculture industry more convenient.

Choline is an officially established essential nutrient and precursor of the neurotransmitter acetylcholine. It is employed as a cholinergic activity marker in the early diagnosis of brain disorders such as Alzheimer’s and Parkinson’s disease. Low levels of choline in diets and biological fluids, such as blood plasma, urine, cerebrospinal and amniotic fluid, could be an indication of neurological disorder, fatty liver disease, neural tube defects and hemorrhagic kidney necrosis. Meanwhile, it is known that choline metabolism involves oxidation, which frees its methyl groups for entrance into single-C metabolism occurring in three phases: choline oxidase, betaine synthesis and transfer of methyl groups to homocysteine. Electrocatalytic detection of choline is of physiological and pathological significance because choline is involved in the physiological processes in the mammalian central and peripheral nervous systems and thus requires a more reliable assay for its determination in biological, food and pharmaceutical samples. Despite the use of several methods for choline determination, the superior sensitivity, high selectivity and fast analysis response time of bioanalytical-based sensors invariably have a comparative advantage over conventional analytical techniques. This review focuses on the electrocatalytic activity of nanomaterials, specifically carbon nanotubes (CNTs), CNT nanocomposites and metal/metal oxide-modified electrodes, towards choline detection using electrochemical sensors (enzyme and non-enzyme based), and various electrochemical techniques. From the survey, the electrochemical performance of the choline sensors investigated, in terms of sensitivity, selectivity and stability, is ascribed to the presence of these nanomaterials.

Mandarin fish (Siniperca chuatsi) is one of the important cultured fish species in China. Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca Chuatsi rhabdovirus (SCRV) have hindered the development of mandarin fish farming industry. Vaccination is the most effective method for control of viral diseases, however viral vaccine production requires the large-scale culture of cells. Herein, a suspension culture system of Chinese perch brain cell (CPB) was developed on Cytodex 1 microcarrier in a stirred bioreactor. Firstly, CPB cells were cultured using Cytodex 1 microcarrier in 125 mL stirring flasks. With the optimum operational parameters, CPB cells grew well, distributed uniformly, and could fully cover the microcarriers. Then, CPB cells were digested with trypsin and expanded step-by-step with different expansion ratios from the 125 mL stirring bottle to a 500 mL stirring bottle, and finally to a 3-L bioreactor. Results showed that with an expansion ratio of 1:3, we achieved a high cell density level (2.25 × 106 cells/mL) with an efficient use of the microcarriers, which also confirmed the data obtained from the 125 mL stirring flask. Moreover, obvious cytopathic effects (CPE) were observed in the suspended CPB cells post-infection with ISKNV and SCRV. This study provided a large-scale culture system of CPB cells for virus vaccine production.

Marine farming of barramundi (Lates calcarifer) in Southeast Asia is currently severely affected by viral diseases. To better understand the biological implications and gene expression response of barramundi in commercial farming conditions during a disease outbreak, the presence of pathogens, comparative RNAseq, and histopathology targeting multiple organs of clinically “sick” and “healthy” juveniles were investigated. Coinfection of scale drop disease virus (SDDV) and L. calcarifer herpes virus (LCHV) were detected in all sampled fish, with higher SDDV viral loads in sick than in healthy fish. Histopathology showed that livers in sick fish often had moderate to severe abnormal fat accumulation (hepatic lipidosis), whereas the predominant pathology in the kidneys shows moderate to severe inflammation and glomerular necrosis. The spleen was the most severely affected organ, with sick fish presenting severe multifocal and coalescing necrosis. Principal component analysis (PC1 and PC2) explained 70.3% of the observed variance and strongly associated the above histopathological findings with SDDV loads and with the sick phenotypes, supporting a primary diagnosis of the fish being impacted by scale drop disease (SDD). Extracted RNA from kidney and spleen of the sick fish were also severely degraded likely due to severe inflammation and tissue necrosis, indicating failure of these organs in advanced stages of SDD. RNAseq of sick vs. healthy barramundi identified 2,810 and 556 differentially expressed genes (DEGs) in the liver and muscle, respectively. Eleven significantly enriched pathways (e.g., phagosome, cytokine-cytokine-receptor interaction, ECM-receptor interaction, neuroactive ligand-receptor interaction, calcium signaling, MAPK, CAMs, etc.) and gene families (e.g., tool-like receptor, TNF, lectin, complement, interleukin, chemokine, MHC, B and T cells, CD molecules, etc.) relevant to homeostasis and innate and adaptive immunity were mostly downregulated in sick fish. These DEGs and pathways, also previously identified in L. calcarifer as general immune responses to other pathogens and environmental stressors, suggest a failure of the clinically sick fish to cope and overcome the systemic inflammatory responses and tissue degeneration caused by SDD.

Scale drop disease virus (SDDV) is one of the most important pathogens that causes scale drop disease (SDD) in Asian sea bass (Lates calcarifer). The outbreaks of this disease are one of the factors causing substantial losses in Asian sea bass aquaculture. In this study, the uracil-DNA glycosylase (UDG)-supplemented cross-priming amplification (UCPA) combined with a colorimetric detection method using the hydroxynaphthol blue (HNB) and lateral flow dipstick (LFD) for detection of SDDV was developed. The UDG was utilized to prevent carryover contamination, and the CPA reactions can be readily observed by HNB and LFD. The CPA primers and probe were designed to target the major capsid protein (MCP) gene of the SDDV. The optimized UCPA conditions were performed at the temperature of 61°C for 60 min. The UCPA assays demonstrated specificity to SDDV without cross-reaction to other tested viruses including red-spotted grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV) and Lates calcarifer herpes virus (LCHV), and other bacterial species commonly found in aquatic animals. The sensitivity of the UCPA-HNB and UCPA-LFD was 100 viral copies/µl and 10 pg of extracted total DNA, which was 10-fold more sensitive than that of conventional PCR. The UCPA-HNB and UCPA-LFD assays could be used to detect the SDDV infection in all 25 confirmed SDDV-infected fish samples. Therefore, the UCPA coupled with HNB and LFD was rapid, simple and effective and might be applied for diagnosis of SDDV infection.

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1β, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

Morphological findings in cases of intoxication are relatively rare in forensic pathology. In this article we report on a 26-year-old man who drank clear fluid from a tequila bottle that was given to him by a friend. Afterwards, the clear fluid was assumed to be smoke fluid containing diethylene glycol (DEG). The man died eight days later. Macroscopic and microscopic examination of the kidneys and the liver at forensic autopsy revealed findings typical of a DEG intoxication. In addition, the clinical course showed the typical triphasic pattern of symptoms. Toxicological analysis confirmed the presence of DEG in both the original smoke fluid and the tequila bottle. In conclusion, death was due to fatal intoxication by DEG. While most DEG intoxications have been mass poisoning incidents attributed to pharmaceutical products, the present case describes an unusual example of a single decedent.

The genus Megalocytivirus (MCV) includes Infectious spleen and kidney necrosis virus (ISKNV) and red seabream iridovirus (RSIV) which are listed by the World Organization for Animal Health (OIE). MCV has a broad host range and can be present as a persistent subclinical infection in some fish. Australia is considered free from MCV and important aquaculture and wild fisheries are at risk. Consequently, biosecurity measures are implemented to ameliorate transmission pathways through imported ornamental fish which include certification of freedom from infection with MCV. Evaluation of practical and cost effective disinfection protocols suitable for recirculating aquaculture facilities is an important aspect of the biosecurity plan to facilitate eradication in the event of early detection of an outbreak. An authentic sample matrix was prepared by in vivo amplification of ISKNV in Murray cod (Maccullochella peelii) and the biological load of a clarified tissue homogenate was standardized by addition of 10% v/v foetal bovine serum. In the absence of a cell culture system for ISKNV, a bioassay was used to assess infectivity after disinfection. A cellulose membrane buffer exchange device was used to remove residual disinfectants before intraperitoneal injection of juvenile Murray cod. Fish were maintained in 100 L aquaria at 23 °C and observed for clinical signs over 14 days. Each bioassay was conducted in duplicate tanks with 6 to 19 fish per tank. A positive assay was defined by an increase in ISKNV DNA quantified by qPCR in any fish from a subsample of challenged fish collected at 7 d or those remaining at 14 d. Negative bioassays were defined by the absence of ISKNV DNA in all fish at the both sampling times. Clinical disease and amplified viral DNA was detected after injection of a dilute positive control, indicating greater analytical sensitivity compared to qPCR (with a detection limit of 100 copies per reaction). Further, the system was used to demonstrate that ISKNV can remain infectious in aquaria without fish for at least 48 h at 25 °C. Effective disinfection measures included: heating to 65 °C for 20 min; pH 3; pH 11; 1% Virkon™; 1000 ppm sodium hypochlorite and benzalkonium chloride at the recommended concentration and contact time. These data can be interpreted to provide effective disinfection protocols for MCV in a wide variety of disease control scenarios.

Megalocytiviruses, particularly red seabream iridovirus, infect a broad range of fish including both freshwater and marine species. Although a limited number of infectious spleen and kidney necrosis virus (ISKNV) strains have been reported in association with mortality events in marine aquaculture species, the potential host range for ISKNV strains, particularly of those that have been detected in ornamental fish, has not been well characterised. There have also been few reports on the susceptibility of euryhaline fish species that could potentially transmit megalocytiviruses between freshwater and marine environments. We found that the euryhaline Australian native percichthyid fish, Australian bass Macquaria novemaculeata, is susceptible experimentally to ISKNV (strain DGIV-10), obtained from a freshwater ornamental fish, dwarf gourami Trichogaster lalius. Australian bass developed clinical disease following direct inoculation and also following cohabitation with infected fish, and were able to transmit DGIV-10 to naïve Murray cod Maccullochella peelii. This study demonstrated the potential for a euryhaline species to become infected with, and transmit, the megalocytivirus ISKNV between fish populations.

There is an increasing number of emerging infectious agents, although not zoonotic, that affect farmed tilapines worldwide. Most recently, the emergence of a new virus, tilapia lake virus (TiLV), has diverted global attention to it while little or no research is being done on the other viruses of possible equal importance. These other viruses have reportedly been associated with relatively high mortalities (20–100%) in several cases of natural disease outbreaks or laboratory challenges. The objective of this review is to highlight viral infections of tilapines that may have been neglected by the scientific community but are also requiring systematic investigation. The viral infections in this review include infectious pancreatic necrosis virus (IPNV, Aquabirnavirus), nervous necrosis virus (VNN, Betanodavirus), tilapia larvae encephalitis virus (TLEV, Herpesvirus), and the Iridovirus infections namely Bohle iridovirus (BIV, Ranavirus) infectious spleen and kidney necrosis virus (ISKNV, Megalocytivirus), Lymphocystivirus and another Iridovirus-like infection. This review summarizes current scientific knowledge on viral infections that affect tilapines and highlights diagnosis methods for disease surveillance and discusses the future visions for disease control.

Infectious spleen and kidney necrosis disease (ISKND) is one of major viral diseases in marine fish species. Here, we isolated infectious spleen and kidney necrosis virus (ISKNV) from diseased barramundi in Thailand and evaluated its pathogenicity with intramuscular challenge in naive barramundi. ISKNV infection in grunt fin (GF) cells was observed within 14 days post inoculation (dpi) by immunofluorescence assay using a monoclonal antibody against red seabream iridovirus (M10). Phylogenetic analysis of the major capsid protein amino acid sequences revealed that two ISKNV isolates belong to genotype I ISKNV. In the challenge test, 35% mortality was observed in fish intramuscularly injected with 10–0.55 TCID50/fish, whereas higher mortalities, 44% and 77%, were observed after injection with 100.55 and 101.55 TCID50/fish, respectively. These results demonstrate dose dependent virulence of ISKNV in barramundi. This study is the first report of ISKNV in farmed barramundi in Thailand. A high susceptibility of barramundi to the infection may suggest an immediate need for control of the emerging disease in barramundi culture including effective vaccine.

Aim: The aim of the study was to determine the prevalence and risk factors of viral nervous necrosis (VNN) disease in tiger grouper cultured in the floating net cage in Bintan district, Indonesia. Materials and Methods: Sampling used multiple stages with ponds as interest units. The selection conducted by systematic random sampling from the entire net cage in Bintan district. The fish samples were selected based on the appearance of clinical signs of infected fish. The risk factors investigated in this study included net cage, technical, and sample fish information, culture, water quality, and feed management. A total of 195 fishes pooled to the 39 net cages and tested using the nested polymerase chain reaction technique to determine the VNN status. The brain and eye processed for histopathology. Results: The prevalence rate of VNN on the net cage was 38% (15/39). The risk factors affecting VNN using bivariate analysis was cleaning the net (χ2=9.80; p=0.002), replacement of net cage (χ2=5.20; p=0.0226), and floating net cage technicians knowledge (χ2=4.13; p=0.042). The variables of positive risk factors affecting VNN by multivariate analysis were the juvenile source and the level of mid-weather changes (technician experience and dissolved oxygen [DO]). Seven variables associated to the VNN outbreak have detected. The positive multiplier factors were the source of juveniles, mid weather changes, technician experience, and the DO, while the negative factors were salinity, mixed feed, and the low weather changes. Histopathologically, the grouper fish showed the brain, eye, and muscle vacuolization and kidney necrosis. Conclusion: It proves that the Bintan waters contaminated by VNN had a prevalence rate of 38% from the total sample based on bivariate method, net cleaning, net replacement, and knowledge on the VNN outbreak. Natural infection of VNN in grouper leads to vacuolization of the brain, the eye, and muscles nearby the eye as well the kidney necrosis.

Megalocytivirus is a genus of piscine viruses that belongs to the Iridoviridae family, and this family comprises red seabream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), and infectious spleen and kidney necrosis virus. RSIV causes high mortality and economic losses in the rock bream (Oplegnathus fasciatus) Korean aquaculture industry. The World Organization for Animal Health’s Office International des Epizooties has provided a manual for RSIV detection. However, detection against TRBIV has not been confirmed. In this study, a multiplex PCR method was established to detect two genotypes of Megalocytivirus, RSIV and TRBIV. New primer pairs were optimized for the PCR reaction. A mixture of one universal and two specific primer pairs could amplify three distinct products targeting three different genes. The sensitivity of the primer pairs was evaluated and results showed a detection limit of 2.0  105 copies for each target gene. Moreover, the primer pairs did not amplify any other viruses. The evaluation of multiplex PCR using 21 RSIV Korean isolates has shown that it can distinguish two genotypes of Megalocytivirus from 21 RSIV Korean isolates. Finally, we describe a new multiplex PCR method to detect different genotypes of Megalocytivirus simultaneously, which makes the diagnosis of viral diseases occurring in the Korean aquaculture industry more convenient.

Ghana’s aquaculture sector is among the recent success stories of fast-growing agricultural value chains in Africa south of the Sahara. The sector has also shown its vulnerability, with the infectious spleen and kidney necrosis virus spreading through tilapia farms in Lake Volta in late 2018. The global COVID-19 human pandemic reached Ghana in early 2020, affecting the sector directly and indirectly. Using a value chain approach, phone interviews were conducted with 369 small-scale fish farmers in six major producing regions, with 12 other value chain actors, and with 423 consumers in the capital, Accra, to assess the impact of COVID-19 on the sector. All value chain actors interviewed reported being affected directly by COVID-19 related restrictions on movement and indirectly by reduced demand for tilapia because of closures in the tourism and hospitality industries, important consumers of fresh tilapia. The crisis has reduced incomes for most actors along the aquaculture value chain and is anticipated to reduce future production. Most fish farmers surveyed were affected by disruptions in input and output markets. Two-thirds of the sample farmers were growing fish and 6 percent were harvesting when the COVID-19 crisis hit. Fifty-four percent of those growing fish experienced difficulties in accessing inputs – mainly fish feeds. Of those harvesting during the crisis, most experienced difficulty in selling their fish mainly because of low demand from buyers, lower tilapia prices, and higher transportation costs than before COVID-19. Income losses among fish farmers, including from other sources, such as crop farming, wage employment, and other own businesses, limits the funds that they have available to finance fish farming operations or to invest in future production capacity. Likewise, reduced incomes and purchasing power of consumers is causing a sharp decline in demand for fish.

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.

In June 2020, an atypical fatal outbreak in a Brazilian Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. Phylogenetic analysis indicated that the virus belongs to the Clade 1 of ISKNV. This viral pathogen is associated with high mortality in the early stages of cultured Nile tilapia in the United States, Thailand and Ghana; however, until now, there have been no reports from ISKNV affecting cultured fish in Brazil. Additionally, in 14 out of 23 sampled fish, Streptococcus agalactiae, Edwardsiella tarda or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms and represents a new challenge to the aquaculture sector in the country.

SummaryIn June 2020, an atypical fatal outbreak in the Nile tilapia farm was investigated. Twenty-three animals were collected and different tissues were used for bacterial isolation, histopathological and electron microscopic examination, and viral detection using molecular methods. A large number of megalocytes were observed in the histopathological analysis of several tissues. Icosahedral virions, with a diameter of approximately 160 nm, were visualized inside the megalocytes through transmission electron microscopy of the spleen tissue. The virions were confirmed to be infectious spleen and kidney necrosis virus (ISKNV) through PCR and sequencing analyses of the fish samples. This viral pathogen is fatal in the early stages of Nile tilapia farms in the USA, Thailand, and Ghana; however, until now, there have been no reports from Brazil. In 14 of the sampled fish, Streptococcus agalactiae, Edwardsiella tarda, or Aeromonas hydrophila infections were also detected. This is the first report of fatal ISKNV infections in the Brazilian Nile tilapia fish farms.

Infectious spleen and kidney necrosis virus (ISKNV), a highly infectious and lethal pathogen of mandarin fish (Siniperca chuatsi), has resulted in major aquaculture losses over the past few decades. A lack of understanding of the regulatory mechanism of viral infection has impeded the development of effective measures to control ISKNV infection. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate the processes of viral infection and are involved in various physiological processes in vivo. In this study, miRNAs involved in the response of mandarin fish to ISKNV infection were identified at several time points following infection through Illumina high-throughput sequencing. A total of 434 (27 known and 407 novel) miRNAs were identified in mandarin fish; 26 (26 up-regulated), 38 (30 up-regulated, 8 down-regulated), and 18 (14 up-regulated and 4 down-regulated) differentially expressed miRNAs (DEMs) were identified at 12, 24, and 48 h post-infection (hpi), respectively. The putative target genes of DEMs were all significantly enriched in host immune response or signaling pathways, such as autophagy, endocytosis, phosphatidylinositol signaling system, Salmonella infection, cell cycle, protein export, and protein processing in endoplasmic reticulum pathway at 12 and 24 hpi and in vascular endothelial growth factor, phosphatidylinositol signaling system and lysosome at 48 hpi. Further analysis indicated that the targeted mRNAs of miRNAs were involved in innate immunity; autophagy and endocytosis might play an important role in immune regulation within 24 hpi with ISKNV. Although the number of DEMs was lowest at 48 hpi, they might still be involved in immunity through the above pathway. We also constructed a miRNA–mRNA targeting network. Generally, the findings of this study provide novel information that advance our understanding of the regulatory mechanisms of miRNA during ISKNV infection in mandarin fish as well as the development of new approaches for preventing ISKNV infection.

Largemouth bass (Micropterus salmoides) is among the most important cultured fish species in China and various viral diseases have greatly affected the largemouth bass industry. We established a permanent cell line from the heart of M. salmoides (MSH) that has been subcultured over 70 times and showed optimal growth at 27 °C using Medium 199 supplemented with 10% fetal bovine serum. MSH cells consisted predominantly of fibroblast-like cells and their karyotypes revealed diploid numbers for most cells at passage 60 with 2n = 64. Sequencing of cytochrome oxidase I and 16S rRNA genes confirmed that MSH cells were derived from M. salmoides. The cell line was tested for Mycoplasma contamination and found to be negative. MSH cells were successfully transfected with a GFP reporter gene indicating that these cells can be utilized for gene expression studies. The MSH cells showed susceptibility to M. salmoides rhabdovirus (MSRV), Largemouth bass ranavirus (LMBV), Infectious spleen and kidney necrosis virus(ISKNV), Spring viremia of carp virus (SVCV), Tilapia lake virus (TiLV) and Grass carp reovirus genotype I (GCRV-I) but were resistant to nervous necrosis virus (NNV). PCR assay and electron microscopy further confirmed that all the tested viruses except NNV can replicate in MSH cells. The replication efficiency of MSRV, LMBV, ISKNV, SVCV, TiLV and GCRV-I ranged from 107.24 to 108.82 TCID50/mL. In addition, type I interferon genes were induced with only NNV infection. In conclusion, we successfully generated an immortal cell line from the heart tissue of M. salmoides and the newly established MHS cell line was highly susceptible to many fish viruses that can be useful for future genetic, virological and immunological studies.